This R21 application addresses complementary important questions about a novel therapeutic modality for perinatal neuroprotection. Although therapeutic hypothermia reduces death or disability after perinatal asphyxia, over 40% of treated infants have poor outcomes. There is an urgent need for more effective thera- pies for perinatal asphyxia and a greater understanding of factors that influence the efficacy of therapeutic cooling. The polyunsaturated fatty acid docosahexaenoic acid (DHA) has intrinsic neuroprotective properties and is under investigation as an oral supplement for pregnant women in several settings. The goal of this proposal, submitted in response to PA-10-069, is to determine the impact of DHA on the neuroprotective efficacy of therapeutic hypothermia in a well-characterized neonatal rodent model of hypoxic-ischemic brain injury. Our laboratory has over 20 years of experience with this model, elicited by unilateral carotid artery ligation and exposure to 8% oxygen in 7 day old (P7) rats. Of particular importance for this proposal, we developed protocols for evaluation of interactions between moderate hypothermia and other therapies that incorporate neuropathological and functional outcome measures. In preliminary studies, DHA was administered parenterally, rather than orally, to neonatal (P7) rats; DHA had neuroprotective properties. This finding provided compelling proof of principle that systemic fatty acid composition influenced susceptibility to neonatal hypoxic-ischemic brain injury. Moreover, our most recent data demonstrate that a single injection of DHA increased the neuroprotective efficacy of subsequent brief moderate hypothermia. We are excited about the potential to translate these findings to clinical studies. Essential prerequisites include: (i) evaluate interactions between DHA supplementation and hypothermia in hypoxic-ischemic neuropathology; (ii) determine the extent to which chronic maternal or acute neonatal DHA supplementation, hypothermia, or both, change brain DHA composition before and after neonatal hypoxia-ischemia, (iii) determine if plasma and/or red blood cell DHA composition reflects neonatal brain DHA composition; (iv) determine effect of DHA, hypothermia, or both, on hypoxia-ischemia-induced fatty acid peroxidation. Aim 1 will evaluate the impact of a range of acute DHA doses or a chronic maternal DHA regimen in P7 rats that undergo hypoxic-ischemic lesioning and hypothermia to identify both optimal combination therapies and potential deleterious interactions. Aim 2 will determine the impact of selected DHA+cooling combination therapies on brain DHA composition. All experiments will be performed on the background of a clinically relevant high fat maternal diet. Results of these studies will provide novel insights about the impact of manipulations of fatty acids on susceptibility to hypoxic-ischemic brain injury, and may also identify unforeseen risks of combination therapy. These studies represent an essential building block for future clinical studies to determine the impact of dietary fatty acids on neonatal brain integrity and susceptibility to perinatal hypoxic-ischemic injury.